Eugenol-Functionalized Bovine Bone Substitutes for Potential Osteogenic Applications

Abstract The study explored the role of eugenol-functionalized bovine bone substitutes in mitigating oxidative stress and promoting osteogenesis in vitro. Bovine bones were subjected to decellularization employing physicochemical and enzymatic procedures to prepare decellularized bovine bone scaffolds (DCC). Eugenol-chitosan functionalized DCC (ECF-DCC) bone substitutes were fabricated by ball-milling DCC scaffolds, spray-drying eugenol-chitosan particulates, integrating β-TCP, and lyophilizing the homogenized composites. ECF-DCC were comprehensively assessed by SEM-EDS, RAMAN spectroscopy, and osteoblast-based viability, apoptosis, reactive oxygen species (ROS) generation, osteogenic differentiation [alkaline phosphatase (ALP), mineralization], real-time PCR, and Western blotting. RAMAN spectroscopy confirmed successful eugenol functionalization of DCC substitutes, evidenced by characteristic aromatic C═C and C–O vibrational peaks alongside preserved phosphate signatures. SEM–EDS revealed rough substitutes in 500–750 μm size with comparable elemental profiles. hFOB 1.19 cells were viable across all concentrations, with no significant increase in apoptosis, although a low-level necrosis was observed at 1000 μg/mL. ECF-DCC significantly reduced ROS generation compared to DCC. Gene expression analysis showed that ECF-DCC markedly reduced key proinflammatory cytokines including IL-6, IL-1β, and TNF-α. Both formulations enhanced osteogenic differentiation, with ECF-DCC inducing superior upregulation of RUNX2, OC, Col1, and BMP-7 at mRNA and protein levels. ECF-DCC substitutes also showed highest ALP activity and calcium mineralization, confirming their enhanced osteoinductive potential. Eugenol-chitosan functionalization enhanced the biological performance of DCC substitutes by reducing oxidative stress and inflammatory responses while significantly promoting osteogenic differentiation, highlighting potential use as improved osteoinductive bone substitutes.